Alcoholic liver disease (ALD) is a chronic condition that affects up to 2 million people in the US, resulting in morbidity and mortality and significant health care expenditures. ALD is characterized by fat accumulation inside liver cells that can lead to inflammation, irreversible cirrhosis and subsequent liver failure. The molecular mechanisms involved in ALD induction are not well understood and there are no effective treatments. This application focuses on the role of the endocannabinoid (EC) signaling pathway in liver development and embryonic and adult models of alcohol-induced hepatic steatosis. A chemical screen conducted in our laboratory revealed this pathway as a positive regulator of liver growth and maturation during development. EC signaling also has important roles in regulating liver lipogenesis and fibrogenesis during chronic liver disease. We will use the zebrafish for our studies, which has advantages as a model organism because of in vivo imaging, time resolution, and chemical screening methods not available or feasible in rodent models. Importantly, zebrafish display disease morphology and pathophysiology that is similar to mammals. We hypothesize that EC signaling impacts cellular metabolism and cell fate during both development and alcohol-induced hepatic steatosis, and these studies can reveal novel drug targets and therapeutic strategies for treating liver damage caused by ALD. Our first aim is to characterize the mechanism of EC signaling during embryonic liver development and embryonic alcohol-induced hepatic steatosis. We have recently generated zebrafish mutants for the two cannabinoid receptors, cnr1 and cnr2, using transcription activator-like effector nucleases (TALEN), a novel technique recently optimized for zebrafish. We will use these mutants and chemical modulators of the EC pathway to characterize liver growth, morphology, and physiology during liver development and assess steatosis in an embryonic model of alcohol-induced hepatic injury after EC pathway modulation. Furthermore, we will elucidate the role of the ghrelin signaling pathway as a collaborator of EC signaling during steatosis induction. Our second aim is to determine the impact of EC signaling on multiple cell types in the liver using two zebrafish models of ALD. We will establish a physiologically relevant adult model of ALD for use in addition to the existing embryonic model. We will then utilize cnr1 and cnr2 mutants and chemical modulators of the EC signaling pathway to uncover histological changes and identify alterations in hepatic stellate cells, liver macrophages, fat metabolism, and biliary tree morphology after alcohol exposure in zebrafish embryos and adults. We then plan to conduct a chemical screen to discover additional compounds or pathways that impact EC signaling during ALD using co-treatment of alcohol with a library of known bioactive compounds. These studies will not only reveal the mechanisms of EC signaling during ALD, but also seek to identify novel therapeutic targets and drugs for the treatment of patients with ALD.